PROJECT SUMMARY Peripheral nerve injuries are life-altering events that can have devastating physical and psychosocial consequences for patients. Functional recovery after nerve injury requires proper communication between a regenerating nerve and the end-target muscle at the neuromuscular junction (NMJ). While much focus of neural regeneration research has been on the nerve itself, the goal of this proposal is to elucidate the molecular mechanisms that drive axonal reinnervation of the NMJ and the response of a special type of glial cell that resides at the NMJ, called terminal Schwann cells (tSCs), after nerve injury. tSCs are non-myelinating SCs that contribute to the formation, maintenance, and regeneration of the synapse, and are especially dynamic after nerve injury. In response to nerve injury, tSCs extend processes that eventually guide regenerating axons to their targets. Although tSC process extension after nerve injury promotes neural regeneration, very little is known about tSCs and the mechanisms that drive this response. Interestingly, Schwann cells (SCs) in a nerve injury site also direct regenerating axons, similar to how tSCs direct regenerating axons at the NMJ, and the mechanisms by which SCs within a nerve injury site guide axons were recently discovered and led to the hypotheses in the current proposal. The goal of Aim 1a is to determine that nerve injury induces regions of hypoxia at the NMJ and ensuing upregulation of HIF-1? (Hypoxia Inducible Factor-1?) target genes. After mice undergo peroneal nerve injury and repair, we will analyze the NMJ of the extensor digitorum longus (EDL) muscles for expression of HIF-1? and 10 known target genes of HIF-1? using qRT-PCR, Western blot, and immunofluorescence. In Aim 1b, the importance of HIF-1? on tSC process extension and eventually axonal reinnervation of the NMJ will be tested by knocking down HIF-1? with local injection of HIF-1? shRNA into the EDL muscle after peroneal nerve injury and repair. Confocal microscopy will evaluate tSC process extension and axonal reinnervation of the NMJ. Macrophage-induced angiogenesis is a well-described process that occurs not only in neural regeneration, but also in other disease states such as wound healing and cancer. In Aim 2, different types of transgenic mice will be used to test the importance of macrophage recruitment (Ccr2-null mice which are unable to recruit macrophages to sites of injury) and macrophage-induced angiogenesis (Vegfafl/flLysmCre mice which lack Vegfa in macrophages) on reinnervation of the NMJ after nerve injury. NMJ reinnervation will be assessed via confocal microscopy and functional recovery will be analyzed by muscle force testing. By defining the molecular mechanisms that drive axonal reinnervation of the NMJ, these studies will provide us with a more comprehensive understanding of recovery after nerve injury. This work will also deepen our understanding of tSC response after injury and lay the foundation for improving outcomes for millions of individuals who suffer from peripheral nerve injuries.